A large number of processes have been described for converting starch to a monosaccharide, glucose. Glucose has value in itself, and also as a precursor for other saccharides such as fructose. Glucose may also be fermented to ethanol or other fermentation products.
The ability of alpha-amylase to hydrolyze raw (unpasted) starch and produce water soluble oligosaccharides has been known since the early 1900's, Reichert, E. T., Publication of the Carnegie Institution at Washington, No. 173, Part 1 (1913). Subsequently, other enzymes have been found that hydrolyze unpasted starch, most notably glucoamylase, which converts granular starch directly to glucose, Evers, A. D. et al., Die Starke 23 (1971), p. 16. The ability of an enzyme to hydrolyze granular starch is associated with the enzyme's ability to be adsorbed on the starch, Walker, G. J. et al., Biochemical Journal 86 (1963), p. 452. The extent of hydrolysis is related to the specific type of starch (corn, potato, wheat, etc.); and its physical condition (granule size; granules intact, fractured, swollen, abraded, etc.), the specific enzyme type and source; the starch and enzyme concentration; the temperature; the pH; and other factors, (Jones, C. R., Cereal Chemistry, 17 (1940), p. 133; Sandstedt, R. M. et al., Journal of the Japanese Society of Starch Science, Vol. 17 (1965), p. 215). The particular effects of these variables are generally only empirically understood.
Several processes for the commercial, low temperature, enzymatic solubilization of granular starch have been proposed, U.S. Pat. Nos. 2,583,451 issued Jan. 22, 1952 to Wallerstein et al; 3,922,196 issued Nov. 25, 1975 to Leach et al; 3,922,197 issued Nov. 25, 1975 to Leach et al; 3,922,198 issued Nov. 25, 1975 to Kuske et al; 3,922,199 issued November 1975 to Hebeda et al; 3,922,200 issued Nov. 25, 1975 to Walon et al and 3,922,201 issued Nov. 25, 1975 to Hebeda et al. Generally, an alpha-amylase is used at pH 4 to 6 at 40.degree. C. to 75.degree. C. to produce a low D. E. syrup. Simultaneously or subsequently, a saccharifying enzyme, such as fungal alpha-amylase, beta-amylase, or glucoamylase may be used at a suitable pH and temperature to produce the desired hydrolysis. The major advantage of such procedures are that they eliminate the high temperature cooking step at 100.degree. C. to 150.degree. C. in presently used syrup processes (U.S. Pat. No. 3,783,100 issued Jan. 1, 1974 to R. F. Larson et al and U.S. Pat. No. 3,875,140 issued Apr. 1, 1975 to Barker et al). Important disadvantages are increased enzyme costs, incomplete solubilization of starch resulting in the need for subsequent starch recycle, mud (fiber, protein and fat) separation problems, and microbial contamination problems.
Recently, it has been discovered that certain fungi, particularly species of the genus Humicola secrete a mixture of enzymes, including enzymes with glucoamylase activity, which efficiently hydrolyze raw (unpasted) starch granules. The ability to hydrolyze starch in granular form is referred to herein as raw starch hydrolyzing (RSH) activity. Enzyme preparations having, in either crude or more refined forms, RSH activity will be referred to herein as RSH enzyme preparations.
RSH preparation obtained from species of the genus Humicola, particularly the species Humicola grisea var. thermoidea, which is hereinafter referred to as Humicola RSH enzyme preparation, has the following characteristics. The Humicola RSH enzyme preparation hydrolyzes raw or granular starch, including straight- and branch-chained starches, and hydrolyzes the starch substantially entirely to glucose. This enzyme preparation is characterized by including a glucoamylase enzyme (EC 3.2.1.3) having an isoelectric point higher than pH 8.0 and a proteinaceous material having glucoamylase-potentiating activity which, in cooperation with the glucoamylase, catalyzes the hyrolysis of the granular starch. Said enzyme preparation is further characterized in that the glucoamylase fraction adsorbs on carboxymethyl cellulose, whereas a fraction containing material exhibiting potentiating activity, or "potentiating factor", is not adsorbed by carboxymethyl cellulose. The Humicola RSH preparation has the ability, for example, to hydrolyze granular starch in a 15% starch solids suspension in water to a solution of saccharides of at least 97% by weight glucose, dry substance basis (d.s.b.), with essentially no starch residue in the absence of debranching enzyme or added alpha-amylase when the hydrolysis is carried out at a pH of between about 5.0 and 7.0, the optimum pH range, and at a temperature of 55.degree. C.
Although certain RSH enzyme preparations will substantially completely hydrolyze granular starch slurries at about 15 weight percent starch, dry solids (d.s.) to produce a high percentage glucose syrup, they generally do not completely solubilize starch in slurries of higher concentration within a reasonable time or provide sufficiently high percentages of glucose.
The ability to hydrolyze and saccharify starch that is raw or only partially pasted represents an important energy savings. Pasting is generally carried out at temperatures of 100.degree. C. to 130.degree. C. and upwards, therefore requiring significant input of thermal energy. RSH enzyme preparations, on the other hand, catalyze starch saccharification efficiently at relatively low temperatures (e.g., at 65.degree. C. or less) and there is no need to heat the starch at any time to higher temperatures. However, starch slurries that are to be hydrolyzed with RSH enzyme preparations may be initially swelled at somewhat higher temperatures (e.g., 65.degree. C. to 100.degree. C.) to hasten the hydrolyzing process. Such a swelling procedure may be considered a partial solubilization and pasting step; nevertheless, starch subjected to these temperatures for short periods of time remains substantially in granular form.
It would be desirable for several reasons to use RSH enzyme preparations to solubilize and saccharify starch slurried at higher solids levels (e.g., between 20 and 60 weight percent starch, and more particularly between about 25 and about 40 weight percent starch, d.s.) than the 15% d.s. level that may generally be hydrolyzed by certain RSH enzyme preparations in a one-step reaction. Higher starch content slurries require less processing reactor tank space. The resulting saccharide solution or syrup is more highly concentrated, and thus, less energy is required for removing water therefrom to further concentrate or dry the same. When slurries higher than 15% solids are hydrolyzed with RSH enzyme preparations, a more concentrated saccharide solution is typically produced; however, in a reaction conducted for a reasonable period of time, a substantial amount of the starch remains insoluble, and unless this remaining starch can also be solubilized or otherwise used, the unsolubilized starch represents a substantial economic loss.
When high starch concentration slurries are enzymatically hydrolyzed with RSH enzyme preparations, hydrolysis generally proceeds at an appreciable rate only for a certain time period, typically about 48 hours. After this, the rate of hydroysis drops off considerably. With glucose being produced at lower rates, it is an inefficient utilization of reactor tank space to allow the reaction to continue. Perhaps more importantly, however, with high glucose concentrations, a significant amount of undesired disaccharide formation occurs and such disaccharides are difficult to hydrolyze. Thus the percent of glucose in the syrup decreases and the percent of higher saccharides increases. Such products are undesirable. For example, if glucose syrup is converted to a high fructose syrup, the higher saccharides do not contribute to the sweetness and may even detract from the sweetness. For these reasons, high solids starch slurries cannot generally be satisfactorily hydrolyzed by RSH enzyme preparations in a single step.
Raw starch, such as raw corn starch, contains in addition to the starch, fiber, protein and fat, which comprise an insoluble sludge or "mud" upon partial solubilization and saccharification of the starch. As the mud builds up in the process system, efficiency of enzymatic starch hydrolysis diminishes. As such, it is not feasible to produce highly concentrated syrups by a simple process of continuously adding additional starch slurry to a reaction vessel as syrup is continuously or periodically removed therefrom. Accordingly, the present invention seeks to solubilize and saccharify high solids granular starch slurries and to recover in solubilized and saccharified form as much starch as possible from the insoluble mud.
It is a primary object of the present invention to provide an economical process which converts a slurry of high solids granular starch substantially completely to glucose (dextrose) at a low temperature. It is a further object of the present invention to provide such a process which facilitates removal of insoluble "mud" (fiber, protein and fat) from the predominantly glucose syrup which is produced.